RESUMEN
OBJECTIVE: To evaluate the clinical accuracy of the IONA® test for aneuploidy screening. METHODS: This was a multicenter blinded study in which plasma samples from pregnant women at increased risk of trisomy 21 underwent cell-free DNA analysis utilizing the IONA test. For each sample, the IONA software generated a likelihood ratio and a maternal age-adjusted probability risk score for trisomies 21, 18 and 13. All results from the IONA test were compared against accepted diagnostic karyotyping. RESULTS: A total of 442 maternal samples were obtained, of which 437 had test results available for analysis and assessment of clinical accuracy. The IONA test had a detection rate of 100% for trisomies 21 (n = 43; 95% CI, 87.98-100%), 18 (n = 10; 95% CI, 58.72-100%) and 13 (n = 5; 95% CI, 35.88-100%) with cut-offs applied to likelihood ratio (cut-off > 1 considered high risk for trisomy) and probability risk score incorporating adjustment for maternal age (cut-off ≥ 1/150 considered high risk for trisomy). The false-positive rate (FPR) was 0% for trisomies 18 and 13 with both analysis outputs. For trisomy 21, a FPR of 0.3% was observed for the likelihood ratio, but became 0% with adjustment for maternal age. CONCLUSION: This study indicates that the IONA test is suitable for trisomy screening in a high-risk screening population. The result-interpretation feature of the IONA software should facilitate wider implementation, particularly in local laboratories, and should be a useful addition to the current screening methods for trisomies 21, 18 and 13.
Asunto(s)
Trastornos de los Cromosomas/diagnóstico , Síndrome de Down/diagnóstico , Pruebas Genéticas/métodos , Pruebas de Detección del Suero Materno/métodos , Trisomía/diagnóstico , Adolescente , Adulto , Trastornos de los Cromosomas/embriología , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 18/genética , Síndrome de Down/embriología , Síndrome de Down/genética , Femenino , Edad Gestacional , Humanos , Cariotipificación , Edad Materna , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Embarazo , Primer Trimestre del Embarazo/sangre , Embarazo de Alto Riesgo/sangre , Embarazo de Alto Riesgo/genética , Método Simple Ciego , Trisomía/genética , Síndrome de la Trisomía 13 , Síndrome de la Trisomía 18 , Adulto JovenRESUMEN
Sertoli and spermatogenic cells establish germ cell- and epithelial stage-dependent networks of cell-cell communication thought to be important for the initiation and maintenance of spermatogenesis. Since gap junctions assemble between Sertoli cells and between Sertoli and spermatogenic cells, it was hypothesized that multiple, unique routes of cell-cell communication may be established, in part, by the assembly of structurally diverse gap junctions from the connexin (Cx) multigene family. Differences in channel structure may support differences in ion or second messenger permeability between cell types. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that 11 Cx mRNAs were present in total RNA from seminiferous tubules and that 10 of the Cx mRNAs were present in polysomes and presumably translated. RT-PCR analyses also showed that the Cx mRNA population varied between different seminiferous tubule cell types. There were 9 Cx mRNAs in germ cells, 8 in Sertoli cells, and 5 in peritubular cells. One of the Cx mRNAs, Cx-50, was detected only in pachytene spermatocytes and round spermatids. Comparisons of the Cx mRNAs present in tubules at different postnatal ages showed that at least 2 Cxs (Cx-33 and Cx-50) accumulated when leptotene-zygotene stages developed. The multiple Cx genes and proteins produced in spermatogenesis may support the assembly of structurally diverse gap junctions.
Asunto(s)
Conexinas/genética , Túbulos Seminíferos/fisiología , Animales , Animales Recién Nacidos , Conexina 26 , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Especificidad de Órganos , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/fisiología , Espermatocitos/fisiología , Proteína beta1 de Unión Comunicante , Proteína alfa-5 de Unión ComunicanteRESUMEN
Immunofluorescence and immunogold electron microscopy were employed to examine the assembly of connexins (Cx) 33, 37, and 43 into testis cell gap junctions in mature and postnatal rats. Cx37 was localized by immunofluorescence to the endothelia of blood vessels in both mature and immature testes and was not further characterized. Only Cx43 assembled into Leydig cell gap junctions, but Cx43 also co-assembled with Cx33 in some Sertoli-Sertoli gap junction plaques within and near Sertoli occluding junctions and on adluminal surfaces Assembly of Sertoli gap junctions appeared to be regulated according to the stage of the seminiferous epithelium since Cx33 (and Cx43) immunoreactivity was strong in Sertoli cells from stages II-VII but weak in stages IX-XIV. During postnatal maturation, assembly of Cx33 into gap junctions was regulated independently of Cx43 assembly. Cx43 was present on Sertoli cells of all tubules from postnatal Day 5 through Day 28. In contrast, Cx33 was not apparent on Sertoli cell surfaces until Day 15 and gradually accumulated in all tubules through Day 28. Between postnatal Days 38 and 43, the immunoreactivities of Cx33 and Cx43 became weak in Sertoli cells containing step 9-14 elongated spermatids. Thus, connexin abundance and gap junction composition in Sertoli cells is regulated during testis maturation and the cycle of the seminiferous epithelium.
Asunto(s)
Conexina 43/biosíntesis , Conexinas/biosíntesis , Uniones Comunicantes/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente Directa , Uniones Comunicantes/ultraestructura , Inmunohistoquímica , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/ultraestructura , Masculino , Microscopía Electrónica , Microscopía Fluorescente , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Epitelio Seminífero/citología , Epitelio Seminífero/metabolismo , Epitelio Seminífero/ultraestructura , Células de Sertoli/ultraestructura , Testículo/crecimiento & desarrollo , Testículo/ultraestructuraRESUMEN
The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
Asunto(s)
Daño del ADN , ADN-Topoisomerasas de Tipo II/biosíntesis , Espermatogénesis/efectos de los fármacos , Tenipósido/toxicidad , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Immunoblotting , Masculino , Espermatogénesis/genética , Xenopus laevisRESUMEN
Immunocytochemical data demonstrate that the distribution of gap junction connexin43 (Cx43) in rodent testes is dependent on cell type, testis maturation, and stage of the mature seminiferous epithelium. Western blotting and indirect immunofluorescence microscopy using anti-peptide antisera to Cx43 revealed abundant Cx43 in rat and mouse testes and mouse TM3 and TM4 cells. Cx43 mRNA was detected in rat testes and mouse TM4 cells by Northern blot analysis. Cx43 was localized by immunogold electron microscopy to gap junctions on Sertoli cells and Leydig cells. A punctate distribution of Cx43 was observed on peritubular cell surfaces following indirect immunofluorescence of detergent-permeabilized tubule segments. In cryosections from testes of immature (to 30 days) rats, and mature rats and mice, Leydig cells showed a punctate surface distribution of Cx43 following indirect immunofluorescence. A diffuse cytoplasmic fluorescence was also seen in spermatocytes and spermatogonia. Cx43 staining associated with Sertoli cells was age- and stage-dependent. Over 90% of the tubules in immature tests (22-30 days) contained Cx43 in the region of Sertoli-Sertoli occluding junctions and in the adluminal compartment. In mature rat testes, however, Cx43 immunostaining was detected in only 60% of 1195 tubule sections where it was abundant proximal to the Sertoli cell occluding junctions. All strongly stained tubules were from stages I-VIII, while negatively stained tubules were at stages IX-XIV. Cx43 immunostaining in mature mouse testes was also stage-dependent with all positive tubules at stages VI-VIII. In contrast to Cx43, Cx26 and Cx32 were detected by immunofluorescence only in the apical regions of the seminiferous epithelia in 90% of tubules from mature rats. Consistent with the observed Cx43 immunostaining, octanol-sensitive in situ dye-coupling was observed between Leydig cells, between peritubular cells and between Sertoli cells, suggesting the occurrence of functional gap junctions in these cell types. These observations provide evidence for extensive gap junction-mediated communication between a variety of testis cell types important to the support of spermatogenesis.
Asunto(s)
Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Testículo/metabolismo , Animales , Conexinas , Inmunohistoquímica , Uniones Intercelulares/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Ratas , Ratas Endogámicas Lew , Epitelio Seminífero/metabolismo , Epitelio Seminífero/ultraestructura , Testículo/crecimiento & desarrollo , Testículo/ultraestructuraRESUMEN
Due to the relative dearth of data regarding somatic cell-germ cell interactions in the testes of non-mammalian chordates, functional homologies between Sertoli cells from diverse organisms have been difficult to assess. However, recent developments in non-mammalian testis cell and organ culture techniques have provided experimental approaches to compare Sertoli cell-germ cell interactions in different vertebrates. Data from in vitro analyses of Sertoli cell-germ cell interactions are presented to suggest that Sertoli cells from rodents and the frog Xenopus laevis have similarities in supporting energy metabolism and glutathione metabolism in spermatogenic cells. Comparative in vitro analyses of Sertoli cell functions should provide further insights into the evolution of cell-cell interactions in the testes.
Asunto(s)
Células de Sertoli/metabolismo , Espermatozoides/metabolismo , Animales , Comunicación Celular , Metabolismo Energético , Glutatión/metabolismo , Humanos , Masculino , Mamíferos , Células de Sertoli/citología , Espermatozoides/citología , Xenopus laevisRESUMEN
Cultured testes and spermatocytes from the frog Xenopus laevis have been incubated (40-42 h) with adriamycin or colcemid followed by quantitation of chromosome aberrations in secondary spermatocytes and quantitation of micronuclei in secondary spermatocytes, early round spermatids, and round spermatids with acrosomal vacuoles (AV) at 18-162 h of culture. Micronucleus frequencies were consistently higher in secondary spermatocytes relative to round spermatids after exposure to either adriamycin or colcemid due to a higher rate of micronucleus formation during meiosis I compared to meiosis II. Also, some of the micronuclei formed during meiosis I did not survive meiosis II to form micronucleated spermatids. Micronucleus formation occurred in 3-7% of secondary spermatocytes with detectable chromosome aberrations, depending upon drug treatment. Thus, the ratio of micronuclei to total chromosome aberrations in secondary spermatocytes was always higher in colcemid-treated cells compared to adriamycin-treated cells following 18- and 42-h treatment periods. Adriamycin induced significant increases in micronuclei in both secondary spermatocytes and spermatids after 162 h of culture, the time for initial pachytene stages to develop into secondary spermatocytes and spermatids. The data show that cultured testes and spermatocytes from Xenopus may be used to quantify specific meiotic chromosome aberrations induced by both clastogens and spindle poisons using either a rapid secondary spermatocyte micronucleus assay or meiotic chromosome analysis.
Asunto(s)
Aberraciones Cromosómicas , Demecolcina/toxicidad , Doxorrubicina/toxicidad , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Espermátides/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Animales , Células Cultivadas , Técnicas de Cultivo , Masculino , Meiosis , Pruebas de Micronúcleos , Espermátides/citología , Espermatocitos/citología , Xenopus laevisRESUMEN
Methods are described for the attachment of isolated spermatocytes to glass slides and the subsequent hypotonic swelling and gradual fixation of the metaphase I and metaphase II cells. The methods minimize cell loss and cell disruption and meiotic metaphase chromosomes become spread within residual cytoplasm thus reducing artefactual chromosome loss. Metaphase II complements from mouse, rat and frog spermatocytes prepared by these procedures had relatively low frequencies of hypoploidy (0.5-1.6%). Bivalent loss was not detected in 916 metaphase I complements. Injection of 0.1 mg/kg demecolcine into mice increased the incidence of metaphase II hypoploidy 8-fold. The hypoploid and hyperploid frequencies here increased equally. The results suggest that the methods described may be useful for the analysis of mechanisms of meiotic aneuploidy including aneuploidy resulting from chromosome loss during meiosis I.
Asunto(s)
Aneuploidia , Cariotipificación/métodos , Espermatocitos/ultraestructura , Animales , Masculino , Meiosis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas Lew , Xenopus laevisRESUMEN
The support of Xenopus laevis spermatogenesis in vitro by different energy-yielding substrates has been investigated. Isolated spermatogenic cells maintained their levels of adenosine-triphosphate for 24 h in serum-free medium containing only amino acids as energy substrates. DL-Aminocarnitine, an inhibitor of carnitine palmitoyltransferase, reduced cell viability 87% during a 15-h culture in the same medium, indicating that beta oxidation of endogenous fatty acids is a significant source of energy when exogenous substrates are unavailable. Isolated spermatocytes developed into spermatids for 7 days in medium supplemented with either pyruvate, oxaloacetate, or lactate, with maximal survival and development at 0.5 mM pyruvate, 2.0 mM oxaloacetate, and 4.0 mM lactate. Few spermatocytes survived more than 3 days in serum-free medium supplemented with only glucose and amino acids as energy substrates. In contrast, glucose-supplemented medium supported spermatocyte differentiation for 14 days in testis fragment culture and 7 days in spermatocyte-Sertoli cell cocultures due to the excretion of lactate and pyruvate by Xenopus Sertoli cells during culture in glucose-supplemented medium. Glucose also enhanced spermatocyte development in medium containing dialyzed, heat-inactivated fetal calf serum. Spermatogenic cells oxidized glucose to CO2 with C1 oxidized 6- to 7-fold more than C6, suggesting that glucose may be metabolized in the hexose monophosphate shunt. The results are discussed in comparison to energy metabolism in mammalian testes and spermatogenic cells.
Asunto(s)
Aminoácidos/farmacología , Metabolismo Energético/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Xenopus laevis/fisiología , Adenosina Trifosfato/metabolismo , Aminoácidos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Medios de Cultivo/farmacología , Glucosa/metabolismo , Lactatos/metabolismo , Masculino , Piruvatos/metabolismo , Células de Sertoli/citología , Células de Sertoli/metabolismo , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Testículo/citología , Testículo/metabolismoRESUMEN
Sertoli cells play a major role in the regulation of spermatogenic cell energy metabolism and differentiation. This study demonstrates that Sertoli cells are essential for the maintenance of spermatogenic cell glutathione (GSH), an important intracellular reductant and detoxicant. Primary spermatocytes and round spermatids isolated from Xenopus laevis contained 1.5 +/- 0.1 mM GSH, but sperm lacked detectable GSH. During a 5-day culture period, isolated spermatocytes and spermatids lost 80% of the initial GSH (t 1/2 = 55 h). The levels of GSH were unaffected by L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of GSH synthesis. Cultures of testicular lobules and spermatocysts (composed of germ cells and Sertoli cells) depleted of interstitial tissue lost only 30% of their initial GSH in 4.5 days; the GSH levels decreased during treatment with BSO. Spermatogenic cells in cultured testes maintained their GSH levels for 7 days by a BSO-sensitive mechanism. These results demonstrate that the intracellular GSH levels of spermatogenic cells are dependent upon germ cell-somatic cell interactions. Spermatogenic cells were shown to possess gamma-glutamyl transpeptidase, glutathione synthetase, 5-oxoprolinase, and gamma-glutamylcysteine synthetase activities. [35S] Cysteine incorporation and distribution as analyzed by high performance liquid chromatography (HPLC) showed that isolated spermatogenic cells are capable of GSH synthesis. The rate of GSH synthesis, however, was insufficient to compensate for GSH turnover. These results demonstrate that production of spermatogenic cell GSH is dependent upon Sertoli cells. To our knowledge, this is the first evidence that interactions between different cell types may be of significance in GSH metabolism.
Asunto(s)
Glutatión/metabolismo , Células de Sertoli/fisiología , Espermatozoides/metabolismo , Animales , Antimetabolitos/farmacología , Butionina Sulfoximina , Comunicación Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Técnicas de Cultivo , Glutatión/biosíntesis , Masculino , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Espermátides/efectos de los fármacos , Espermátides/metabolismo , Espermatocitos/efectos de los fármacos , Espermatocitos/metabolismo , Espermatogénesis , Espermatozoides/efectos de los fármacos , Espermatozoides/crecimiento & desarrollo , Espermatozoides/ultraestructura , Testículo/metabolismo , Testículo/fisiología , Testículo/ultraestructura , Xenopus laevisRESUMEN
Explant cultures of testes from the frog Xenopus laevis have been employed to evaluate the application of testis culture to the routine screening of potential germ cell genotoxicants. Testis explants were incubated with varied concentrations of 3 model mutagens (9,10-dimethyl-1,2-benzanthracene, cyclophosphamide, adriamycin) and solvent controls. Round spermatids were isolated from testes cultured 2-30 days after exposure to each mutagen. The spermatids were then stained with Hoechst 33258 and spermatid micronuclei were scored with a fluorescence microscope. Acute exposure of testes to each mutagen resulted in a dose-dependent increase in spermatid micronuclei that was stage specific and proportional to the length of the exposure period. The assay sensitively detected clastogenic effects by 10(-7) M adriamycin (4-h exposure period) and 10(-6) M cyclophosphamide and dimethylbenzanthracene (24-h exposure). The results demonstrate the feasibility of in vitro approaches to the routine screening and investigation of genotoxicity in the premeiotic S through meiotic division stages of vertebrate spermatogenesis.
Asunto(s)
Pruebas de Mutagenicidad/métodos , Espermátides/ultraestructura , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , 9,10-Dimetil-1,2-benzantraceno/farmacología , Ciclo Celular/efectos de los fármacos , Aberraciones Cromosómicas , Ciclofosfamida/farmacología , Doxorrubicina/farmacología , Masculino , Técnicas de Cultivo de ÓrganosRESUMEN
Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.
Asunto(s)
Polimorfismo Genético , Protaminas/análisis , Espermatozoides/análisis , Xenopus laevis/genética , Animales , Cromatina/análisis , Electroforesis en Gel de Poliacrilamida , Masculino , Protaminas/genéticaRESUMEN
Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. 3H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since 3H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis.
Asunto(s)
Espermatogénesis , Testículo/fisiología , Animales , Medios de Cultivo , Replicación del ADN , Femenino , Fertilización , Masculino , Técnicas de Cultivo de Órganos , Motilidad Espermática , Testículo/citología , Tritio , Xenopus laevisRESUMEN
DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5-100 micrograms/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5-6 micrograms/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6-100 micrograms/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 micrograms/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by "intermediate-type" protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.
Asunto(s)
Bufonidae/fisiología , ADN Superhelicoidal/ultraestructura , Rana catesbeiana/fisiología , Espermatogénesis , Xenopus laevis/fisiología , Animales , Bufonidae/genética , ADN Superhelicoidal/efectos de los fármacos , Eritrocitos/ultraestructura , Etidio/farmacología , Masculino , Rana catesbeiana/genética , Especificidad de la Especie , Espermatozoides/ultraestructura , Xenopus laevis/genéticaRESUMEN
Spermatid/sperm basic chromosomal proteins from 17 species and subspecies of the genus Xenopus (Anura, Pipidae) were compared. Electrophoresis on acetic acid/urea/Triton X-100 polyacrylamide gels revealed that each Xenopus species with a diploid chromosome number of 36, 72, or 108 showed multiple, diverse spermatid/sperm-specific basic chromatin proteins with mobilities greater than the somatic histones. The numbers and mobilities of these proteins were characteristic of each Xenopus species and each subspecies of Xenopus laevis. Cytochemical tests revealed that the sperm basic nuclear proteins of these Xenopus species and subspecies were rich in arginine and lysine and contained more arginine than the nuclear proteins of somatic cells. X. tropicalis (2n = 20) and X. sp. n. (Zaire) displayed spermatid/sperm-specific basic chromatin proteins which migrated within the histone H1 region of acetic acid/urea/Triton X-100 polyacrylamide gels. Cytochemically the sperm nuclei of these species resembled those of somatic cells. These observations suggest that spermatid/sperm basic nuclear proteins can be used as molecular markers for individual species and subspecies of the genus Xenopus.
Asunto(s)
Proteínas Cromosómicas no Histona/análisis , Histonas/análisis , Espermátides/citología , Espermatozoides/citología , Animales , Diploidia , Eritrocitos/análisis , Masculino , Especificidad de la Especie , XenopusRESUMEN
The morphogenesis of sperm nuclei was investigated in six different species or subspecies of the genus Xenopus (Pipidae, Anura). The sequence of nuclear morphogenesis was similar in each species used in this study. Electrophoretic comparison of the basic chromatin proteins from late spermatids and sperm of each species demonstrated that the complements of histones and spermatid-sperm-specific basic proteins were extremely diverse suggesting that shape was not determined by specific basic proteins or mechanisms of histone removal. This conclusion was reinforced by the observation that Xenopus sperm DNA decondensed by 2.0 M NaCl remained contained in residual structures which resembled intact sperm nuclei. These observations suggested that morphogenesis of sperm nuclei is directed by proteins or RNA molecules which are not directly responsible for chromatin condensation.
Asunto(s)
Núcleo Celular/ultraestructura , Espermatozoides/ultraestructura , Xenopus/anatomía & histología , Animales , Cromatina/análisis , ADN , Histonas/análisis , Masculino , Conformación de Ácido Nucleico , Conformación Proteica , Especificidad de la EspecieRESUMEN
For the past three years a team of educational evaluators and health professionals has worked on development of (1) definitions of competence in occupational therapy, clinical dietetics and medical technology, and (2) associated prototype assessment instruments. This study has also involved an effort to develop a research methodology for pursuing similar investigations in other fields. That methodology, the Professional Performance Situation Model, proved appropriate for occupational therapy. A second application with clinical dietetics brough a number of significant modifications which support the view that PPSM is a valid special model for direct patient contact health professions. The third application, with medical technology, is intended to extend PPSM as a general model for competence definition in health professions. This article concentrates on the application of PPSM in clinical dietetics. It is argued that evidence for the feasibility and validity of PPSM is sufficient to justify confidence in the methodology. Completion of the next phase of development promises to support the authors' contention that they have developed a general paradigm for competence definition and assessment in all health professions.